A single-step purification of biologically active recombinant human interleukin-5 from a baculovirus expression system.

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  • Additional Information
    • Source:
      Publisher: Academic Press Country of Publication: United States NLM ID: 9101496 Publication Model: Print Cited Medium: Print ISSN: 1046-5928 (Print) Linking ISSN: 10465928 NLM ISO Abbreviation: Protein Expr Purif Subsets: MEDLINE
    • Publication Information:
      Publication: Orlando, FL : Academic Press
      Original Publication: San Diego : Academic Press, c1990-
    • Subject Terms:
      Baculoviridae/*genetics ; Interleukin-5/*genetics ; Interleukin-5/*isolation & purification; Amino Acid Sequence ; Animals ; Base Sequence ; Cell Line ; DNA, Complementary ; Humans ; Mice ; Molecular Sequence Data ; Receptors, Interleukin/metabolism ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Spodoptera
    • Abstract:
      Recombinant human interleukin-5 (rhIL-5) was expressed in baculovirus-infected insect cells and purified to homogeneity from the culture medium in a single chromatographic step. Beginning with a cDNA encoding the full-length precursor form of human IL-5, including the authentic secretory leader sequence, recombinant baculovirus-infected insect cells expressed high levels of rhIL-5 (5-15 mg/liter culture) of which > 90% was processed to the mature form and secreted into the culture medium. After removing cells by centrifugation, rhIL-5 was purified by first adjusting the culture medium to the calculated pI value of mature IL-5 (pI 7.44) and then passing the conditioned medium through tandem linked anion- and cation-exchange columns. The resulting pass-through fraction contained the rhIL-5 and was devoid of contaminating proteins. An optional hydrophobic-interaction chromatography step effectively concentrated the pure homodimeric N-glycosylated rhIL-5 with a high overall yield (> 90%). N-terminal amino acid sequence determination indicated that cleavage of the human IL-5 leader sequence in insect cells occurred between Ala19 and Ile20. Recombinant human IL-5 prepared by this procedure bound to the high-affinity IL-5 receptor present on an eosinophilic leukemia cell line and elicited a proliferative response in the IL-5-dependent murine B-cell line BCL1. This rapid and simple procedure for the expression and purification of mature rhIL-5 should therefore enable studies requiring large amounts of this cytokine.
    • Accession Number:
      0 (DNA, Complementary)
      0 (Interleukin-5)
      0 (Receptors, Interleukin)
      0 (Recombinant Proteins)
    • Publication Date:
      Date Created: 19950201 Date Completed: 19950629 Latest Revision: 20191031
    • Publication Date:
      20231215
    • Accession Number:
      10.1006/prep.1995.1009
    • Accession Number:
      7756840